Click Chemistry Palmitoylation Assay

Click chemistry palmitoylation assay was conducted imitating the method described by Coleman DT, et al. [15 (link)]. In brief, after the metabolic labeling the palmitoylated proteins with palmitate orthologs alkyne-linked 17-Odya (Cayman Chemical, USA), the cells lysates were further processed through the click reaction between biotin-azide (Cayman Chemical, USA) and alkyne-linked 17-Odya, which make the palmitoylated proteins covalently cross-linked with biotin at the S-palmitoylation sites. The biotinylated protein were pulled down from the cells lysates using streptavidin-agarose mini-column (Sigma, USA), and then were subjected to western blotting using monoclonal antibody recognizing EYFP (Amyjet Scientific, China).